Cellular proliferation in all organisms requires the sequential execution of DNA replication, chromosome segregation, and cell division. To coordinate these activities, cells employ regulatory checkpoints that ensure faithful completion of each step before attempting the next. Although the cell cycle has been well studied in eukaryotes, our understanding of cell cycle networks and regulatory controls in bacteria remains rudimentary. Caulobacter crescentus represents an excellent model for studies of cell cycle control in bacteria as it is genetically tractable, easily synchronized, and exhibits distinct G1, , and G2 phases. C. crescentus exhibits a DNA replication checkpoint, such that when DNA replication is blocked, cell cycle progression halts. Previous work demonstrated that DNA replication initiation somehow promotes activation of CtrA, an essential cell cycle regulator. CtrA is a response regulator of the two-component signaling protein family that, when phosphorylated, acts as a global transcription factor, promoting expression of genes necessary for cell division. In the absence of DNA replication, CtrA is not activated, thus ensuring that cels do not attempt cell division prematurely. However, the molecular mechanism underlying this critical cell cycle checkpoint remains unknown and is the focus of this project. High-throughput candidate and unbiased genetic approaches will be used to identify potential regulators necessary for coupling CtrA regulation to DNA replication. The candidate regulators identified in these screens will be tested alongside one previously identified CtrA regulator for their roles in linking DNA replication to CtrA activation. Genetic and biochemical approaches will be used to distinguish how these regulators promote activation of CtrA in a DNA replication-dependent manner. Thus, the results from this study will provide important insights into the regulatory mechanisms coupling DNA replication to cell cycle progression in bacteria. Studies of the bacterial cell cycle are important as they (1) can reveal regulatory principles conserved in all domains of life and (2) will reveal critical differences that could be exploited to develop new antibiotics.